Resting latently HIV-1–infected cells were treated with Nef inhibitors for 4–6 days. Cells were then cocultured at a 1:1 ratio with autologous HIV-1 peptide–expanded CD8 T cells in medium containing HAART. One day later, an MHC-I blocking antibody, W6/32, was added to the wells to prevent further CD8 T cell–mediated killing, along with IL-15 to induce virus production. Six days later, the frequency of CD4 T cells producing HIV-1 Gag p24 were enumerated via flow cytometry. Shown in A are representative plots of an experiment with CD4 T cells infected with the NL4-3 virus. (B) Paired data between the control and B9-treated latently NL4-3–infected cell cocultures with expanded CD8 T cells are shown, and the summary data with each indicated treatment is shown. (C) Paired results between cocultures of latently NL4-3 ΔNef–infected cells receiving the inhibitor B9 and the DMSO control are shown. (D) Data were normalized to the DMSO control condition and are reported as the Residual Gag⁺ CD4 T cells, the percentage of HIV-1 Gag p24⁺ cells as a fraction of the DMSO control. Paired data between control and B9 cocultures and summary data are shown. Medians shown for B and C. Mean with SEM shown for summary data in D. (B–D) NL4-3 WT: n = 15 B9; n = 14 others. NL4-3 ΔNef: n = 10 B9, n = 8 others. All analyses were performed comparing the control (NT) data to the paired Nef inhibitor data. Wilcoxon matched-pair tests were performed to determine statistical significance in B and C. Paired t tests were performed to determine statistical significance for Residual Gag⁺ cells (%) data in D. Reported P values have been corrected for multiple comparisons using the Benjamini-Hochberg procedure. *P < 0.05, **P < 0.01, ***P < 0.001.